2.1 IFN Therapy for Treatment of Viral Infection
Interferons are extracellular signaling proteins which have many diverse effects, including immune stimulation, tumor inhibition, and cell multiplication reduction. Interferon induces host cell production of many gene products, including the Mx proteins, the 2'-5' oligoadenylate synthetase and RNase L. (reviewed in Sen et al., 1992, J. Biol. Chem. 267: 5017-5020; Sen et al., 1993, Adv. Virus. Res. 42: 57). In addition, PKR, a cAMP-independent serine/threonine kinase, is one of the greater than 30 INF-induced gene products (Meurs et al., 1990, Cell 62: 379-590). These proteins have been shown to have a protective effect against viral infection, thus contributing to the beneficial effects of IFN therapies in treating viral infections.
There are many mechanisms by which IFN-induced gene products provide a protective effect against viral infection. For example, the IFN-induced double-stranded RNA-dependent enzyme, 2'-5' oligoadenylate synthetase activates an RNase that cleaves cellular and viral RNAs, thereby inactivating viral replication, such as picornavirus replication (Kumar et al, 1988, J. Virol. 62: 3175-3181). The IFN-induced double-stranded RNA-dependent protein kinase inhibits virus and host cell protein synthesis by phosphorylating and inactivating the .alpha. subunit of the translational initiation factor eIF-2.alpha.. IFN also has inhibitory viral effects at different stages of the viral life cycle. IFN inhibits uncoating of hepatitis B viral particles, thereby inhibiting viral replication. IFN inhibits the penetration of vesicular stomatitis virus into cells (Whitaker-Dowling et al., 1983, Proc. Natl. Acad. Sci. 80: 1083-1086). IFN also inhibits cell fusion caused by viruses, such as Sendai virus (Tomita et al., 1981, J. Gen. Virol. 55: 289-295).
To counteract the deleterious effects of IFN-induced cellular defense mechanisms, many eukaryotic viruses have evolved mechanisms to block the activity of IFN-induced proteins. A number of viruses produce RNAs which override the IFN-induced host cell shut off of translation, for example, the adenovirus produced VAI RNA molecule, human immunodeficiency virus (HIV) TAR region, and the Epstein-Barr virus produced EBER-1 RNA molecule (Katz et al., 1987, Embo. J. 6: 689-697; McMillan et al., 1995, Virology 213: 413-424; Carroll et al., 1993; Beattie et al., 1995, Virology 210: 254-263; Beattie et al., 1991, Virology 183: 419-422). A number of viruses such as the vaccinia virus K3L protein, which binds to and inhibits IFN-induced PKR protein kinase (Gale Jr. et al., 1996, Mol. Cell. Biol. 16: 4172-4181). Influenza virus, recruit cellular factors, such as P58 which binds to and blocks PKR protein kinase (Lee et al., 1990, Proc. Natl. Acad. Sci. 87: 6208-6212; Lee et al., 1994, Mol. Cell. Biol. 14: 2331-2342).
2.2 Hepatitis C Virus
Hepatitis C virus (HCV) infection is an important clinical problem worldwide. In the United States alone, an estimated four million individuals are chronically infected with HCV. HCV, the major etiologic agent of non-A, non-B hepatitis, is transmitted primarily by transfusion of infected blood and blood products (Cuthbert et al., 1994, Clin. Microbiol. Rev. 7: 505-532; Mansell et al., 1995, Semin. Liver Dis. 15: 15-32). Prior to the introduction of anti-HCV screening in mid-1990, HCV accounted for 80-90% of posttransfusion hepatitis cases in the United States. Currently, injection drug use is probably the most common risk factor for HCV infection, with approximately 80% of this population seropositive for HCV. A high rate of HCV infection is also seen in individuals with bleeding disorders or chronic renal failure, groups that have frequent exposure to blood and blood products.
Acute infection with HCV results in persistent viral replication and progression to chronic hepatitis in approximately 90% of cases. For many patients, chronic HCV infection results in progressive liver damage and the development of cirrhosis. In patients with an aggressive infection, cirrhosis can develop in as little as two years, although a time span of 10-20 years is more typical. In 30-50% of chronic HCV patients, liver damage may progress to the development of hepatocellular carcinoma. In general, hepatocellular carcinoma is a late occurrence and may take greater than 30 years to develop (Bisceglie et al., 1995, Semin. Liver Dis. 15: 64-69). The relative contribution of viral or host factors in determining disease progression is not clear.
HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.5 kb. On the basis of its genome organization and virion properties, HCV has been classified as a separate genus in the family Flaviviridae, a family that also includes pestiviruses and flaviviruses (Alter, 1995, Semin. Liver Dis. 15: 5-14). The viral genome consists of a lengthy 5' untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3' UTR. The 5' UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation. Translation of the HCV genome is initiated by a cap-independent mechanism known as internal ribosome entry. This mechanism involves the binding of ribosomes to an RNA sequence known as the internal ribosome entry site (IRES). An RNA pseudoknot structure has recently been determined to be an essential structural element of the HCV IRES. Although a reliable tissue culture system permissive for HCV replication has yet to be developed, viral proteins have been identified by a variety of techniques, including the use of recombinant vaccinia virus constructs and transcription/translation systems. The polyprotein precursor is cleaved by both host and viral proteases to yield mature viral structural and nonstructural proteins. Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, E1 and E2. HCV also encodes two proteinases, a zinc-dependent metalloproteinase, encoded by the NS2-NS3 region, and a serine proteinase encoded in the NS3 region. These proteinases are required for cleavage of specific regions of the precursor polyprotein into mature peptides. The carboxyl half of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase. The function of the remaining nonstructural proteins, NS4A and NS4B, and that of NS5A (the amino-terminal half of nonstructural protein 5) remain unknown.
Progress in understanding HCV replication and pathogenesis has been severely hampered by the lack of an cell culture system. As a result, effective antiviral strategies against HCV infection have yet to be developed. Treatment with alpha interferon is currently the sole therapy for chronic HCV infection. However, less than 50% of patients have sustained remissions following treatment, with the eradication of HCV (Hoofnagle et al., 1994; Lino et al., 1994, Intervirology 37: 87-100). As discussed above, there is a correlation between HCV genotype and response to interferon therapy (Enomoto et al., 1996, N. Engl. J. Med. 334: 77-81; Enomoto et al., 1995, J. Clin. Invest. 96: 224-230). For example, the response rate in patients infected with HCV-1b is less than 40%. Similar low response rates for patients infected with prototype United States genotype, HCV-1a, have also been reported (Hoofnagle et al., Lino et al, 1994, Intervirology 37: 87-100). In contrast, the response rate of patients infected with HCV genotype-2 is nearly 80% (Fried et al., 1995, Semin. Liver Dis. 15: 82-91).
The amino acid sequence of a discrete region of the NS5A protein of genotype 1b was found to correlate with sensitivity to interferon (Enomoto et al., 1996, Enomoto et al., 1995). This region, termed the interferon sensitivity determining region (ISDR), spans amino acid residues 237 to 276 of NS5A [amino acids 2209 to 2248 of the polyprotein; numbering based on that of HCV-J, an HCV-1b strain for which the complete genomic sequence has been determined (Enomoto et al., 1995). Interferon-resistant strains (i.e., quasispecies that were present prior to, as well as six months post-interferon therapy) possess the same ISDR sequence as that of the prototype HCV-1b strain (Enomoto et al., 1996). In contrast, an interferon-sensitive strains (HCV quasispecies that were present prior to interferon treatment but absent six months post-therapy) multiple amino acid substitutions are found within the ISDR. In this study, patients infected with HCV isolates containing four to 11 amino acid changes within the ISDR showed a complete response to interferon therapy. In contrast, in patients infected with HCV isolates containing the prototype ISDR sequence (and 87% of those infected with HCV isolates containing one to three amino acid changes within the ISDR) no response to therapy was observed. The mechanism by which the ISDR of HCV-1b influences the response to interferon is not known.